Project Summary/Abstract: The protein complexin plays a critical role in the regulation of SNARE mediated synaptic vesicle exocytosis at presynaptic nerve terminals in the brain. Complexin functions both to inhibit spontaneous synaptic vesicle fusion and to enhance synchronized fusion. The central helix domain of complexin is required for both its inhibitory and its facilitatory functions, but additional regions of the protein, in particular the C- terminal domain (CTD), which follows the central helix and the accessory helix, which immediately precedes the central helix, are also required for complexin's full inhibitory function. Our objective is to understand the molecular basis for the functional roles of the CTD and the accessory helix. The CTD of complexin interacts with phospholipid membranes, and this interaction serves to localize complexin to synaptic vesicles in vivo and is required for complexin's inhibitory function. Two motifs within the CTD act to both localize complexin specifically to highly curved synaptic vesicle membranes and to activate complexin's inhibitory activity only when bound to synaptic vesicles, but important questions remain regarding the mechanisms by which membrane-binding contributes to complexin's inhibitory function. Specifically, the structural basis for the curvature-dependent interactions of the two CTD motifs with membranes remain unclear. In addition, many complexins feature a C-terminal CAAX box motif that leads to farnesylation, and the membrane-binding properties of farnesylated complexins have not been explored. This proposal aims at filling key gaps in our knowledge of how the structural features of membrane-bound complexin relate to the function of the protein. To do this, two specific aims will be pursued. (1) To characterize the structural basis for the membrane curvature-dependent structural transition of the amphipathic helix motif of complexin's CTD, to assess the role of membrane curvature dependent helix formation in regulating complexin's inhibitory function and to assess the influence of this transition on membrane binding affinity and kinetics. (2) To characterize the structural basis for membrane interactions by both farnesylated and unmodified C-terminal motifs in complexin's CTD, and to asses the functional implications of these interactions. The accessory helix of complexin also contributes significantly to the inhibition of neurotransmitter release. Recent results suggest that the accessory helix acts to nucleate and stabilize helical structure in the central helix, and thereby facilitates the interactions of the central helix with SNARE proteins. In a third specific aim (3) we will test this hypothesis by directly characterizing alterations in the helicity of the accessory and central helices, measuring how such alterations influence SNARE binding in vitro, and correlating these measurements with effects on complexin's inhibitory function in vivo. Together these three aims will serve to elucidate the molecular basis for the roles of complexin's CTD and accessory helix in the inhibition of neurotransmitter release.